MARINE BIOLOGY
Marine Biology 54, 185-194 (1979)
9 by Springer-Verlag 1979
Acetate Incorporation into the Lipids of the Anemone Anthopleura elegantissima and Its Associated Zooxanthellae R.S. Blanquet l, J.C. Nevenzel 2 and A.A. Benson2 [ Department of Biology, Georgetown University; 37th & O Sts, NW, Washington, D.C. 20057, USA and 2Marine Biological Research Division, Scripps Institution of Oceanography, University of California, San Diego; La Jolla, California 92093, USA
Abstract
containing zooxanthellae, as well as isolated zooxanthellae, incubated with acetate-1-14C under both light and dark conditions readily incorporate r a d i o a c t i v i t y into their total lipid pools. In both c a s e s , the specific activity was greatly increased in the light. Dark-incubated anemones and isolated zooxanthellae incorporate activity p r e d o m i n a n t l y into polar lipid; the remainder being present principally in the triglyceride moiety. L i g h t - i n c u b a t e d organisms, however, show a dramatic r e d i s t r i b u t i o n of isotope towards greatly increased triglyceride and wax ester incorporation, with a concomitant drop in polar lipid. Under light conditions, 70 to 75% of the radioactivity found in the fatty acids of the total zooxanthellae lipid was present in hexadecanoic (16:0) and octadecenoic (18:1) fatty acids. These are also the two major fatty acids by mass. Octadecanoic acid (18:O) is less than 5% by mass. Isotope incorporation patterns suggest that octadecenoic acids arise by elongation of hexadecenoic acids and that this conversion is blocked in the dark. Isotope incorporation patterns for anemones suggest that fatty acids, p r i m a r i l y in the form of saturated or monoenoic fatty acids, are translocated from algal to animal cells. No activity was found in either octadecadienoic (18:2) or o c t a d e c a t r i e n o i c (18:3) acids. The significance of these data is discussed. Anthopleura elegantissima
I ntroduction
Lipids are integral, structural components of all cell membranes and also provide a major source of energy throughout the animal kingdom. Anthozoans, such as sea anemones and corals, have been shown to have a high lipid content and contain appreciable lipid energy reserves, principally in the form of triglycerides and wax esters (Bergman et al., 1956; Rouser et al., 1963; Hooper and Ackman, 1971; Hill, 1976) which may vary markedly with season (Hill, 1976). Anthozoans are also rich in s p e c i e s which contain within their tissues, intracellular algal symbionts called zooxanthellae. This is true for all scleractinian, or reef-building, corals and numerous anemones and soft corals. It is now well established that zooxanthellae can translocate soluble, photosynthetically derived, compounds to the cells of their animal hosts and thus supply them with a significant amount of utilizable organic material at low m e t a b o l i c cost
(Muscatine and Hand, 1959; Goreau and Goreau, 1960; yon Holt and von Holt, 1968; Muscatine and Cernichiari, 1969; Smith et al., 1969; Taylor, 1969, 1973; Trench, 1970, 1971a,b,c,; Lewis and Smith, i97i; Young et el., 1971; Muscatine et al., 1972; Chalker and Taylor, 1975). This recycling of carbon has been viewed as especially significant in corals which live in nutrient-poor, tropical s e a s . Although lipids play crucial roles in cell integrity and energy production, there has been, until recently, little evidence that zooxanthellae provide their hosts with lipid by-products. Experiments in which cnidarians were exposed to radiolabeled NaH14CO 3 have shown that labeled carbohydrate, primarily in the form of glycerol and glucose, appears to be the main product liberated from the zooxanthellae (Muscatine and Cernichiari, 1969; Lewis and Smith, 1971; Trench, 1971a,b). In some c a s e s where animal lipids were found to be labeled, the radioactivity was found to
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r e s i d e e x c l u s i v e l y in the g l y c e r o l m o i ety of d e a c y l a t e d c o m p o u n d s (von H o l t and von Holt, 1968; M u s c a t i n e and Cernichiari, 1969; Trench, 1971a). Y o u n g et al. (1971) r e p o r t e d s i g n i f i c a n t incorp o r a t i o n of l a b e l e d c a r b o n into the lipid of the coral Pocillopora capitata w h i c h c o n s i s t e d m a i n l y of the w a x ester, c e t y l p a l m i t a t e . The p r o p o s e d s t a r t i n g p o i n t was a s s u m e d to be c a r b o h y d r a t e in the form of g l y c e r o l which, after b e i n g c o n v e r t e d to A c e t y l Co A, was then synt h e s i z e d to p a l m i t a t e . Thus, lipid app e a r e d to be s y n t h e s i z e d in the coral p r i m a r i l y from c a r b o h y d r a t e p r o d u c t s . Recently, however, P a t t o n et al. (1977) p r o v i d e d e v i d e n c e s u p p o r t i n g the d i r e c t t r a n s f e r of l a b e l e d f a t t y acids f r o m z o o x a n t h e l l a e to a n i m a l cells. T h e y prop o s e d that a c e t a t e p r o d u c e d by a n i m a l cells was c o n v e r t e d to fatty acids w i t h in the z o o x a n t h e l l a e using p h o t o s y n t h e tically derived adenosine triphosphate (ATP) and r e d u c i n g power. T h e s e fatty acids w e r e then r e t u r n e d to the host cells w h e r e they w e r e c o n v e r t e d to trig l y c e r i d e and w a x ester. This s c h e m e w o u l d then supply the host w i t h lipid e n e r g y at lower m e t a b o l i c cost. It is the p u r p o s e of this study to c o n f i r m and to e x p l o r e the u t i l i z a t i o n of a c e t a t e for fatty acid b i o s y n t h e s i s in w h o l e a n i m a l s and i s o l a t e d zooxant h e l l a e using the sea a n e m o n e Anthopleura el egan ti ssima.
Materials and Methods
S p e c i m e n s of Anthopleura elegantissima w e r e c o l l e c t e d from rocks e x p o s e d at low tide in the v i c i n i t y of S c r i p p s O c e a n o g r a p h i c Institute, C a l i f o r n i a , USA. On r e t u r n i n g to the l a b o r a t o r y , each a n e m o n e was c a r e f u l l y c l e a n e d of a d h e r i n g bits of sand, rock and shell, b l o t t e d dry and w e i g h e d . Care was taken to e n s u r e that w a t e r c o n t a i n e d in the g a s t r o v a s c u l a r c a v i t y was e x p e l l e d . Dry w e i g h t d e t e r m i n a t i o n s w e r e m a d e using two m e t h o d s . The first was to dry a n e m o n e s to c o n s t a n t w e i g h t in an o v e n at 6OOC. The s e c o n d was to e x p r e s s dry w e i g h t as total i n s o l u b l e m a t e r i a l a f t e r h o m o g e n a t e p r e c i p i t a t i o n by t r i c h l o r a c e t i c acid (TCA) . This was a c c o m p l i s h e d by h o m o g e n i z i n g each i n d i v i d u a l in a k n o w n v o l u m e of d e i o n i z e d w a t e r for 2 m i n at h i g h speed in a glass blender. An e q u a l v o l u m e of 20% T C A was then added to y i e l d a 10% T C A p r e c i p i t a b l e fraction. This f r a c t i o n i n c l u d e d p a r t i c u l a t e c e l l u l a r m a t e r i a l plus s u b s t a n c e s prec i p i t a t e d by the TCA. The i n s o l u b l e mat e r i a l was c o l l e c t e d by c e n t r i f u g a t i o n , and then r e s u s p e n d e d and w a s h e d four
times in d e i o n i z e d w a t e r to r e m o v e excess T C A and salts. This m a t e r i a l was th~] d r i e d to c o n s t a n t w e i g h t at 60~ V a l u e S are e x p r e s s e d as p e r c e n t of initial wet weight. Isolation of Zooxanthellae
Z o o x a n t h e l l a e w e r e l i b e r a t e d from anemone t i s s u e by h o m o g e n a t i o n at h i g h s p e e d in a glass b l e n d e r for 2 min. L a r g e particles w e r e a l l o w e d to settle by g r a v i t y and the s u p e r n a t a n t , c o n t a i n i n g the zoox a n t h e l l a e , was decanted. The s e t t l e d m a t e r i a l was r e s u s p e n d e d and the p r o c e s s was r e p e a t e d three times. The r e s u l t i n g s u p e r n a t a n t s c o n t a i n i n g a d d i t i o n a l zoox a n t h e l l a e w e r e a d d e d to the first. Zoox a n t h e l l a e w e r e p e l l e t e d by c e n t r i f u g a tion and w e r e w a s h e d three s u c c e s s i v e times in f i l t e r e d sea w a t e r after carefully r e m o v i n g (between each wash) the upper, w h i t i s h layer w h i c h c o n s i s t e d of c e l l u l a r debris. M i c r o s c o p i c e x a m i n a t i o n of the final algal p e l l e t s h o w e d a h i g h ly p u r i f i e d algal s u s p e n s i o n . No c h l o r o phytes, r e p o r t e d to be p r e s e n t in some p o p u l a t i o n s of Anthopleura elegantissima (Muscatine, 1971), w e r e ever seen. To e s t i m a t e the a m o u n t of z o o x a n t h e l lae p r e s e n t in each anemone, all steps w e r e taken to m a x i m i z e the z o o x a n t h e l l a e y i e l d o b t a i n e d by the above p r o c e d u r e . F i n a l a l g a l p e l l e t s w e r e r e s u s p e n d e d in a k n o w n v o l u m e of sea water. Two equal a l i q u o t s w e r e removed, p e l l e t e d , and w e i g h e d in a t a r e d vessel. T h e s e samples w e r e s u b s e q u e n t l y d r i e d to c o n s t a n t w e i g h t at 60~ and r e w e i g h e d . Cell counts (10 replicates) m a d e on e a c h sample e n a b l e d an e s t i m a t i o n of total algal wet and dry weight. This was then exp r e s s e d as p e r c e n t ~f a p p r o p r i a t e w h o l e a n i m a l weight. Lipid Extraction and Fractionation
In s t u d i e s d e s i g n e d to d e t e r m i n e the total lipid c o n t e n t of Anthopleura elegantissima, lipid was q u a n t i t a t i v e l y e x t r a c t e d by the m e t h o d of B l i g h and D y e r (1959). T o t a l lipid s a m p l e s of a n e m o n e s and isolated z o o x a n t h e l l a e u s e d for i s o t o p e and chromatographic analyses were obtained by two s u c c e s s i v e , 24 h, e x t r a c t i o n s in c h l o r o f o r m : m e t h a n o l (2:1, v/v). A n e m ones w e r e m i n c e d p r i o r to e x t r a c t i o n . The e x t r a c t e d lipids w e r e filtered, d r i e d via r o t a r y e v a p o r a t i o n , and s t o r e d u n d e r n i t r o g e n at -15oc. Chromatography and Radioisotope Studies
Q u a n t i t a t i v e d e t e r m i n a t i o n s of lipid c l a s s e s was a c h i e v e d by c h r o m a t o g r a p h y on U n i s i l p a c k e d c o l u m n s a c c o r d i n g to
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the m e t h o d of N e v e n z e l et al. (1965). W a x and s ~ e r o l e s t e r s w e r e e l u t e d w i t h I% d i e t h y i e t h e r in p e t r o l e u m ether (boiling p o i n t 60 ~ to 70oc) and t r i g l y c e r i d e s in 5% d i e t h y l e t h e r . M o r e p o l a r lipids w e r e e l u t e d w i t h 100% d i e t h y l e t h e r f o l l o w e d by a b s o l u t e m e t h a n o l . P r o d u c t s e l u t e d by these s o l v e n t s w e r e c o m b i n e d and coll e c t i v e l y c a l l e d p o l a r lipid. This fraction c o n s i s t e d of a m i x t u r e of sterols, p h o s p h o l i p i d , and p e r h a p s some free acids and a l c o h o l s . C o m p o n e n t s of t o t a l lipid s a m p l e s were q u a l i t a t i v e l y i d e n t i f i e d via thinlayer c h r o m a t o g r a p h y (TLC) and a p p r o p r i ate s t a n d a r d s . T o t a l lipid c o m p o n e n t s w e r e s e p a r a t e d and run on s i l i c a gel G a n a l y t i c a l p l a t e s (0.25 m m t h i c k n e s s , EM l a b o r a t o r i e s ) u s i n g two s o l v e n t systems: t h a t of M a n g o l d (1965) and a d o u b l e ~ o l v e n t s y s t e m of F r e e m a n and W e s t (1966). In b o t h systems, w a x ester and s t e r o l e s t e r s m i g r a t e as a s i n g l e spot. These, h o w e v e r , c o u l d s u b s e q u e n t l y be s e p a r a t e d u s i n g a s o l v e n t s y s t e m cons i s t i n g of h e x a n e : b e n z e n e (60:40, v/v) run in two d i m e n s i o n s (Hill, 1976). Spots w e r e d e t e c t e d by i o d i n e v a p o r or u n d e r u l t r a - v i o l e t light a f t e r s p r a y i n g w i t h O . 0 0 1 2 % R h o d a m i n e 6G in d i s t i l l e d water. P r i o r to s e p a r a t i o n , 10 ~i aliquots of each s a m p l e w e r e s p o t t e d and w e i g h e d in t r i p l i c a t e on a C a h n m i c r o b a l a n c e , M o d e l 4700. Total lipid samples obtained from a n e m o n e s or i s o l a t e d z o o x a n t h e l l a e exp o s e d to 1 4 C - l a b e l e d a c e t a t e w e r e rout i n e l y f r a c t i o n a t e d by the s o l v e n t system of F r e e m a n and W e s t (1966). Spots c o r r e s p o n d i n g to p o l a r lipid, sterols, t r i g l y c e r i d e s and w a x / s t e r o l e s t e r s w e r e always d e t e c t e d in a n e m o n e or z o o x a n t h e l l a e e x t r a c t s but the w a x / s t e r o l ester spot was a l w a y s f a i n t in the latter. A r e a s c o r r e s p o n d i n g to each w e r e s c r a p e d f r o m the p l a t e and p l a c e d in s c i n t i l l a t i o n v i a l s to w h i c h 10 ml of A q u a s o l was then added. A r e a s b e t w e e n each d e t e c t e d spot w e r e also s c r a p e d and counted. Alt h o u g h no spots w e r e u s u a l l y d e t e c t e d in these areas, they w e r e a r b i t r a r i l y lab e l e d for the lipid c o m p o n e n t k n o w n to m i g r a t e in that area. Thus, the e n t i r e d e v e l o p e d area for e a c h s a m p l e was counted. A c t i v i t i e s w e r e m e a s u r e d on a Beckman LSSOOO liquid scintillation c o u n t e r and r e c o r d e d u s i n g a T e x a s Ins t r u m e n t S i l e n t 700 e l e c t r o n i c d a t a terminal.
Isotope Incorporation Studies
A n e m o n e s used for these s t u d i e s w e r e c o l l e c t e d and t r e a t e d as p r e v i o u s l y described. A n e m o n e s w e r e a l l o w e d to s e t t l e
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and a t t a c h to the b o t t o m of 50 ml beakers o v e r n i g h t in the light at 18oc. S u b s e q u e n t l y , 20 ~Ci of a c e t a t e - 1 - 1 4 C was i n j e c t e d into the g a s t r o v a s c u l a r cavity, and the a n e m o n e s w e r e a l l o w e d to i n c u b a t e u n d i s t u r b e d for 3 or 12 h. A n e m o n e s w e r e then removed, rinsed, and p l a c e d in f i l t e r e d sea w a t e r for two s u c c e s s i v e 15 m i n p e r i o d s to a l l o w for f l u s h i n g of the g a s t r o v a s c u l a r cavity. Dark-incubated individuals were treated in a s i m i l a r m a n n e r , but w e r e p l a c e d in the dark for 30 min p r i o r to a d m i n i s t r a tion of the isotope. Z o o x a n t h e l l a e s u s p e n s i o n s w e r e prep a r e d as p r e v i o u s l y d e s c r i b e d . Cell counts w e r e taken p r i o r to the a d d i t i o n of i s o t o p e and r a n g e d from 80 to 110 x 104 cells ml -I . A l g a l s u s p e n s i o n s (25 ml) w e r e a l l o w e d to r e m a i n in the light or the d a r k for 30 m i n p r i o r to the addition of 20 ~Ci of a c e t a t e - 1 - 1 4 C . S u s p e n sions w e r e then a l l o w e d to i n c u b a t e for 3 to 12 h. A l g a l cells w e r e s u b s e q u e n t ly w a s h e d three times in 25 ml a l i q u o t s of fresh, f i l t e r e d sea w a t e r to r e m o v e n o n - i n c o r p o r a t e d isotope. Fatty Acid Methylation
M e t h y l a t i o n of fatty acids from lipid samples obtained from acetate-incorporation s t u d i e s was a c c o m p l i s h e d by h e a t i n g s a m p l e s d i s s o l v e d in 0.5 N s o d i u m m e t h y l a t e in m e t h a n o l d i l u t e d w i t h b e n z e n e 3:1 (v/v) in a b o i l i n g w a t e r b a t h for 3 min. G l a c i a l a c e t i c acid was used to n e u t r a l i z e the s o l u t i o n a f t e r heating. The m e t h y l e s t e r s w e r e e x t r a c t e d into h e x a n e and w a s h e d twice w i t h equal v o i umes of d i s t i l l e d water, or u n t i l the w a t e r was a p p r o x i m a t e l y n e u t r a l w i t h r e s p e c t to pH. The h e x a n e was then rem o v e d by e v a p o r a t i o n and the m e t h y l esters s t o r e d u n d e r N 2 at -15oc. P r i o r to gas c h r o m a t o g r a p h i c a n a l y s i s , m e t h y l esters w e r e p u r i f i e d via TLC on silica gel G p l a t e s and d e v e l o p e d in a s o l v e n t s y s t e m of b e n z e n e : h e x a n e (30:70, v/v). In this solvent, m e t h y l e s t e r s have an Rf v a l u e of a p p r o x i m a t e l y 0.25. Gas c h r o m a t o g r a p h y was c a r r i e d out on two i n s t r u m e n t s . I n i t i a l a n a l y s e s w e r e run u s i n g a P a c k a r d Gas C h r o m a t o g r a p h S e r i e s 7400 t o g e t h e r w i t h a P a c k a r d Prop o r t i o n a l R a d i o a c t i v i t y C o u n t e r 894. Mass and r a d i o a c t i v i t y p e a k s w e r e recorded simultaneously. Separations were a c c o m p l i s h e d i s o t h e r m a l l y at 150 ~ or 185~ u s i n g a 6 ft (1.82 m), 4 m m innerd i a m e t e r glass c o l u m n p a c k e d w i t h 10% S i l a r I0C on 1 0 0 / 1 2 0 m e s h (24 ~ ) Gas C h r o m e Q. F a t t y acid m e t h y l e s t e r s w e r e i d e n t i f i e d by c o c h r o m a t o g r a p h y and by c o m p a r i s o n of their r e l a t i v e r e t e n t i o n times w i t h a u t h e n t i c s t a n d a r d s .
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I n i t i a l i d e n t i f i c a t i o n of f a t t y acids was c o n f i r m e d b e f o r e and after a r g e n t a tion c h r o m a t o g r a p h y (Dunn and Robson, 1965) on a P e r k i n - E l m e r M o d e l 3920 B Gas Chromatograph together with a radioact i v i t y d e t e c t o r i d e n t i c a l to the one u s e d initially. Two c o l u m n s w e r e employed: a 6 ft (1.82 m), 2 ram innerdiameter, s t a i n l e s s steel, 8% OV17 on 80/1OO m e s h (28 ~um) C h r o m o s o r b W - H P column run i s o t h e r m a l l y at 22OOC and a 6 ft, 2 m m i n n e r - d i a m e t e r , s t a i n l e s s steel 10% DEGS c o l u m n (80/100 mesh) run i s o t h e r m a l ly at 185oc. A r e a s for mass and r a d i o a c t i v i t y peaks were d e t e r m i n e d by t r i a n g u l a t i o n . R e l a t i v e s p e c i f i c a c t i v i t y for each fatty acid was d e t e r m i n e d by d i v i d i n g the area of the m a s s p e a k by t h a t of the r a d i o a c t i v i t y peak.
Results
E x p r e s s i o n s of total lipid c o n t e n t in o r g a n i s m s are m o s t o f t e n g i v e n as a percent of e i t h e r w e t or dry w e i g h t . In marine i n v e r t e b r a t e s , p a r t i c u l a r l y those such as c n i d a r i a n s w h i c h have high water content, v a l u e s c i t e d may v a r y considerably, d e p e n d i n g on the m e t h o d employed. In Anthopleura elegantissima, w h o l e i n d i v i d u a l s , d r i e d to c o n s t a n t w e i g h t at 60oc, d e m o n s t r a t e a b o u t twice the percent dry w e i g h t as that d e t e r m i n e d by T C A p r e c i p i t a t i o n (Table I). The f o r m e r d e t e r m i n a t i o n i n c l u d e s i n o r g a n i c salt and low m o l e c u l a r w e i g h t c o m p o u n d s w h i l e the latter, to a larger degree, does not. By e i t h e r method, A. elegantissima have a h i g h lipid c o n t e n t (Table 2). B a s e d on w e t weight, v a l u e s c i t e d for total lipid are in g o o d a g r e e m e n t w i t h those c i t e d in an e a r l i e r study of A. e!egantissima (1.56%) by R o u s e r et al. (1963). V a l u e s r e p o r t e d for other anemones, c o l l e c t e d at v a r i o u s w a t e r t e m p e r atures and seasons, are I .3% for Metridium dianthus (Hooper and Ackman, 1971) and 2.4% (Mason, 1972) and 0.7 to 5.2% (Hill, 1976) for M. senile. On a dry w e i g h t basis, the total lipid v a l u e s g i v e n here for A. elegantissima are s o m e w h a t lower than the 33% r e p o r t e d by B e r g m a n et al. (1956) for Condylactis gigantea. E s t i m a t i o n s of algal m a t e r i a l in w h o l e i n d i v i d u a l s are a b o u t 14% w e t w e i g h t or 8% dry w e i g h t (Table I). T h e s e v a l u e s s h o u l d be r e g a r d e d as r o u g h app r o x i m a t i o n s . A l t h o u g h care was taken to m a x i m i z e the y i e l d of z o o x a n t h e l ! a e for each anemone, an unknown, but p r o b a b l y s i g n i f i c a n t , a m o u n t of a l g a e was lost d u r i n g the i s o l a t i o n p r o c e d u r e s . Thus, the v a l u e s cited may be r e g a r d e d as being on the low side.
Whole anemones contain about equal a m o u n t s of w a x e s t e r s and t r i g l y c e r i d e , w h i c h t o g e t h e r a c c o u n t for a b o u t 18% of the total e x t r a c t a b l e lipid. Levels of t r i g l y c e r i d e and wax esters for zooxant h e l l a e are a b o u t 6 and 7%, r e s p e c t i v e ly (Table 3). As z o o x a n t h e l l a e a c c o u n t for only 8% of the total a n i m a l dry weight, the c o n t r i b u t i o n of these algal lipids to w h o l e a n i m a l p o o l s w o u l d be small.
Lipid Classes
The s p e c i f i c r a d i o a c t i v i t y of the total lipid d e r i v e d from w h o l e a n e m o n e s or z o o x a n t h e l l a e l i g h t - i n c u b a t e d w i t h acet a t e - 1 - 1 4 C was always g r e a t e r than in d a r k - i n c u b a t e d o r g a n i s m s , thus d e m o n strating increased acetate utilization d u r i n g p e r i o d s of p h o t o s y n t h e s i s (Fig. I). For i s o l a t e d z o o x a n t h e l l a e , the s p e c i f i c a c t i v i t y of the total lipid i s o l a t e d after light i n c u b a t i o n for 12 h was alm o s t ten times that for d a r k - i n c u b a t e d algae. The v a l u e for s i m i l a r l y t r e a t e d w h o l e a n e m o n e s was a b o u t two times as high. Of p a r t i c u l a r i n t e r e s t is the dis-
Table i. Anthopleura elegantissima. Weight determinations. Values are means ~ standard deviation; numbers in parentheses indicate numbers of determinations Whole anemone (% dry weight) Oven-dried TCA precipitate at 6oOc
Zooxanthellae (% of whole anemone) Wet weight Dry weight (oven, 60 ~
25.3~1.8(8)
13.6•
12.9~1.9(ii)
7.7•
Table 2. Anthopleura elegantissima. Total lipid determinations. Five anemones per group were used. SD: standard deviation Group
Wet weight (g)
Total lipid (g)
Total lipid (% of whole anemone) Wet weight Dry weight Dry weight (6OOC) (TCA)
1 2 3 4
10.33 9.50 8.59 7.04
O.29 O.31 0.28 0.25
2.73 3.29 3.24 3.62 3.22•
Mean~SD
11.17 13.14 12.96 14.47 12.9491.36
Table 3. Anthopleura elegantissima. Lipid-class determinations (% total Lipid). Numbers in parentheses indicate number of determinations Sample
~
o
Zooxanthellae(2)
5.6
6.5
87.9
Whole anemone(3)
9.0
9.3
81.7
acontains some sterol ester. bcontains sterols, phospholipids, chlorophylls, galacto- and phospholipids.
21.51 25.23 24.88 27.67 24.82•
R.S. Blanquet et al.: Lipogenesis in Anthopleura and Zooxanthellae
ACETATE-1-140 I N C O R P O R A T I O N Anemone
ACETATE-1-14C I N C O R P O R A T I O N Zooxanthellae
Spee. Act. L/D -4.7 Total Lipid
9O
189
Spee, A c t . L - 15 Total L i p i d "/'D '
SP ec. Act. L. / ~ - 9 3 Total Lipid / ~
Spec. Act. L//D_2. 2 Total Lipid
90
7O
70
>
~ 50 < 0 F,
&
~ 30 s
1o
PL
ST
FA TG 3 - HOUR
WE
PL
ST
FA TG 12 - H O U R
WE
PL
ST
FA TG 3-HOUR
WE
PL
ST
FA TG 12-HOUR
WE
Fig. i. Anthopleura elegantissima. Acetate-1-14C incorporation into various lipid classes of isolated zooxanthellae and of whole anemones during 3 and 12 h incubation periods. Incorporation into light- and dark-incubated organisms is indicated by open and shaded bars, respectively. Specific activity is expressed as ratio of light(L)- to dark(D)-incubated organisms. PL: polar lipid; ST: sterols; FA: fatty acids (may include alcohols and diglyceride) ; TG: triglyceride; WE: wax ester (includes sterol ester)
t r i b u t i o n of r a d i o a c t i v i t y a m o n g the v a r i o u s lipid c l a s s e s (Fig. I). D a r k - i n c u b a t e d a n e m o n e s , as w e l l as z o o x a n t h e l l a e , i n c o r p o r a t e a c t i v i t y pred o m i n a n t l y into p o l a r lipid, w i t h v a l u e s r a n g i n g f r o m 75 to 90%. The r e m a i n d e r is found p r i m a r i l y in t r i g l y c e r i d e s . L i g h t i n c u b a t e d a n e m o n e s show a d r a m a t i c redistribution towards increased triglyceride and w a x e s t e r i n c o r p o r a t i o n . S i n c e the w a x e s t e r f r a c t i o n c o u n t e d c o n t a i n e d detectable sterol ester components, these w e r e s e p a r a t e d via TLC and c o u n t e d s e p a r a t e l y . At 3 h in the light, 95% of the r a d i o a c t i v i t y was f o u n d in wax esters c o m p a r e d to 71% in the dark. As the a m o u n t of w a x e s t e r in z o o x a n t h e l l a e is low and shows little i s o t o p e i n c o r p o r a tion, the i n c r e a s e d a c t i v i t y in a n i m a l w a x e s t e r is p r o b a b l y due to s y n t h e s i s from l a b e l e d algal p r e c u r s o r s such as free acids or a l c o h o l s . Z o o x a n t h e l l a e i n c u b a t e d in the light also show a large i n c r e a s e in t r i g l y c eride r a d i o a c t i v i t y . In c o n t r a s t to whole anemones, incorporation exceeded that for p o l a r lipid, p a r t i c u l a r l y after 12 h, w h e n 70% of the total a c t i v i t y was found in this f r a c t i o n . A l t h o u g h no i o d i n e - d e t e c t a b l e spots w e r e u s u a l l y seen b e t w e e n the s t e r o l and t r i g l y c e r i d e spots s e p a r a t e d via TLC w i t h the s o l v e n t s y s t e m of F r e e m a n and W e s t (1966), this area was s c r a p e d f r o m the plates, counted, and a r b i t r a r i l y la-
b e l e d free fatty acid (FA) in Fig. I. In a d d i t i o n to free f a t t y acids, however, a l c o h o l s and d i g l y c e r i d e s are also k n o w n to m i g r a t e in this area (Hill, 1976). W h i l e no c o m p o n e n t s w e r e v i s u a l i z e d , s i g n i f i c a n t a c t i v i t y was n o t e d for b o t h light- and d a r k - i n c u b a t e d z o o x a n t h e l l a e . I n c r e a s e d a c t i v i t y in this r e g i o n for light v e r s u s dark w h o l e a n e m o n e s m a y r e p r e s e n t a t r a n s l o c a t i o n of fatty acids from algae to a n e m o n e d u r i n g p h o t o s y n thetic periods, since a n i m a l d a r k inc o r p o r a t i o n was v e r y low. The i n c o r p o r a tion of a c e t a t e into s t e r o l s was v e r y low for all e x p e r i m e n t a l c o n d i t i o n s .
Fatty Acids
The fatty acid c o m p o s i t i o n and r e l a t i v e r a d i o a c t i v i t y of the total lipid ext r a c t e d from z o o x a n t h e l l a e and w h o l e a n e m o n e s i n c u b a t e d in a c e t a t e - 1 - 1 4 C is g i v e n in T a b l e s 4 and 5, r e s p e c t i v e l y . H e x a d e c a n o i c (16:0) and o c t a d e c e n o i c (18:1) are the two m a j o r fatty acids by m a s s o b t a i n e d from z o o x a n t h e l l a e . T h e i r a c t i v e b i o s y n t h e s i s d u r i n g p e r i o d s of p h o t o s y n t h e s i s is r e f l e c t e d in the h i g h levels of a c e t a t e i n c o r p o r a t i o n , w h i c h a c c o u n t e d for 70 to 75% of the total r a d i o a c t i v i t y . Of note is the low content of o c t a d e c a n o i c (stearic, 18:O) acid w h i c h m e a s u r e d only a b o u t 5% by mass. A l g a l s p e c i e s over a w i d e r a n g e of
190
R.S. Blanquet etal.: Lipogenesis in A n t h o p l e u r a and Zooxanthellae
Table 4. Anthopleura elegantissima. Fatty acid composition and 14C incorporation of iso]ated zooxanthellae incubated in acetate-l-14C. Data is expressed as averaged percentages of total fatty acid mass and radioactivity detected. Relative specific activity (RSA) = area of mass p e a k : a r e a of radioactivity peak. L: lightincubated; D: dark-incubated. N is total number of determinations; ave: average Fatty acid a
Isolated zoox~nthellae 3 b incubation (N=2) Mass (ave) 14C incorporation L D L D RSA L
(ave) RSA D
12 h incubation (N=3) Mass (ave) 14C incorporation L D L D RSA L
(ave) RSA D
14:0
2.8
4.1
3.0
13.4
2.7
5.2
1.8
4.5
3.6
5.6
3.7
1.i
16:0
18.4
18.3
43.8
12.8
5.9
i.i
25.2
19.6
40.7
28.9
4.9
i .3
16:1
4.9
7.4
18.9
53.7
9.4
11.7
4.3
4.2
7.0
56.3
5.7
11.4
18:0
3.0
6.2
1.5
2.8
4,8
18:1
19.3
19.7
31.5
13.9
4.0
1.i
24.4
21.O
32.2
18:2
1.5
3.5
2.6
1.6
2.7
1.2
1.5
1.3
3.9
3.9
0.7
1.3
18:3
2.5
4.0
1.7
1.8
22:6
13.3
13.1
13.5
14.1
aFatty acids are designated
by carbon chain
length;number
4.1 8.5
3.8
0.4
of double bonds.
Table 5. Anthopleura elegantissima. Fatty acid composition and 14C incorporation of whole anemones incubated in acetate-l-14c. Data is expressed as averaged percentages of total fatty acid mass and radioactivity detected. Relative specific activity (RSA) - area of mass p e a k : a r e a of radioactivity peak. L: light-incubated; D: dark-incubated. N is total number of determinations; ave: average Fatty acid a
Whole anemones 3 h incubation (N=2) Mass (ave) 14C incorporation L D L D RSA L
(ave) RSA D
12 h incubation (N=3) Mass (ave) 14C incorporation L D L D RSA L 0.5
1.0
0.2
10.8
12.1
33.7
3.4
3.7
8.3
14:0
i.i
1.3
-
-
16:0
18.6
17.9
34.9
32.9
2.4
16:1
6.0
5.8
]2.3
-
2.8
18:0
11.3
13.0
8.2
51.8
1.3
0.5
9.9
8.7
]2.3
18:1
24.6
18.1
42.7
16.0
2.4
0.i
19.4
18.2
43.7
18:2
5.0
5.0
-
-
1.5
2.1
18:3
2.0
3.0
-
-
6.3
1.1
20:4
13.0
14.1
-
-
12.3
12.6
20:5
16.0
15.3
-
-
17.4
i?.2
22:6
2.8
2.0
-
-
1.8
2.7
aFatty acids are designated
by carbon chain length:number
f a m i l i e s have b e e n shown to c o n s i s t e n t l y have such low levels of this acid, alt h o u g h oleic a c i d levels can v a r y cons i d e r a b l y (Wood, 1974). P a t t o n et al. (1977), however, have r e p o r t e d that s t e a r i c acid c o m p r i s e d over 35% of the fatty acids i s o l a t e d f r o m the t r i g l y c eride and p h o s p h o l i p i d f r a c t i o n s of Pocillopora capitata z o o x a n t h e l l a e . No a c t i v i t y was d e t e c t a b l e in s t e a r i c acid after 3 h of a c e t a t e i n c u b a t i o n . A f t e r 12 h, however, the d e t e c t a b l e act i v i t y w i t h r e l a t i v e l y h i g h s p e c i f i c act i v i t y w o u l d s u g g e s t de n o v o s y n t h e s i s from a c e t a t e v i a the m i t o c h o n d r i a l system. S i g n i f i c a n t levels of a c t i v i t y w e r e found in h e x a d e c e n o i c acid (16:1). Inc o r p o r a t i o n of i s o t o p e into o c t a d e c a d i e n o i c (18:2) and o c t a d e c a t r i e n o i c (18:3) fatty acids was low, even after 12 h e x p o s u r e s . The low 18:O fatty acid i n c o r p o r a t i o n level s u g g e s t s that the h i g h 18:1 acid c o n t e n t arises via its s y n t h e s i s from a 16:1 fatty acid. This is f u r t h e r en-
54.2
0.7
(ave) RSA D
0.3
0.6 45.8
0.5
0.4
1.2
of double bonds.
h a n c e d by the s t r i k i n g r e d i s t r i b u t i o n of i s o t o p e i n c o r p o r a t i o n for the 16:O, 16:1 and 18:1 fatty acids in d a r k - i n c u b a t e d z o o x a n t h e l l a e . I n c o r p o r a t i o n into palm i t o l e i c acid (16:1) is d r a m a t i c a l l y increased, w i t h c o n c o m i t a n t drops in palm i t i c and oleic acid. The e x t r e m e l y h i g h r e l a t i v e s p e c i f i c a c t i v i t y of p a l m i t o l e ic acid s u p p o r t s its de novo s y n t h e s i s from a c e t a t e r a t h e r than its f o r m a t i o n by d e s a t u r a t i o n of p r e e x i s t i n g p o o l s of u n l a b e l e d p a l m i t a t e . In addition, the d e c r e a s e d a c t i v i t y for oleic acid could r e s u l t from a b l o c k in the c o n v e r s i o n of p a l m i t o l e i c acid to o l e i c in the d a r k w h i c h does n o t e x i s t in the light. Inc o r p o r a t i o n of label into p o l y u n s a t u rated fatty acids (18:2, 18:3) is enh a n c e d d u r i n g p e r i o d s of light, but rem a i n s low even after 12 h. No d e t e c t a b l e i n c o r p o r a t i o n of these acids a p p e a r s in w h o l e a n i m a l lipids. For w h o l e anemones, 16:0, 18:0, 18:1 and two p o l y u n s a t u r a t e d acids t e n t a t i v e ly i d e n t i f i e d as 20:4 and 20:5 are the
R.S. Blanquet et al.: Lipogenesis in Anthopleura and Zooxanthellae
principal fatty acids by mass. With the exception of a higher 18:1 fatty acid content found in this study, the fatty acid profile given for Anthopleura elegantissima (Table 5) is in good agreement with that found for Metridium senile (Mason, 1972). High eicosatetraenoic (20:4) and e i c o s a p e n t a e n o i c (20:5) acid content has been d e m o n s t r a t e d for several anemones of both warm- and cold-water habitats (Bergman, 1956; Hooper and Ackman, 1971; Mason, 1972). Principal incorporation in the light appears in 16:O and 18:1 acids, but 16:1 and 18:O possess considerable activity. D a r k - i n c u b a t e d anemones, however, seem to only synthesize saturated fatty acids, with levels of incorporation into 18:O acid rising sharply. A l t h o u g h 16:1 fatty acids are highly labeled in d a r k - i n c u b a t e d zooxanthellae (Table 4), no label was detectable in this fatty acid for darkincubated whole anemones (Table 5). A possible explanation may be that since 16:1 is a minor component of the total algal lipid fatty acid pool and the zooxanthellae mass accounts for only 8% of the total animal dry weight, when combined with the animal pool, the activity of the 16:1 fatty acid moieties was below the instrument level of detection.
Discussion
Recently, Patton et al. (1977) proposed a carbon cycle in coral whereby the acetate g e n e r a t e d by animal m e t a b o l i s m is utilized by endozoic algae to manufacture lipid which is then returned to the host. Our studies, using the zooxanthellae-containing anemone Anthopleura elegantissima, provide support for this scheme. For both whole anemones and isolated zooxanthellae, labeled acetate is readily incorporated into fatty acids. In each case, this process is strongly enhanced by light (Fig. I), presumably via the excess ATP and reducing potential generated via photosynthesis. Lightinduced synthesis of fatty acids occurs in other unicellular algae, as well as higher plants, and has been extensively studied (Eppley et al., 1963; N a k a m u r a and Yamada, 1975a,b; Stumpf, 1975). The rate of synthesis, the degree of unsaturation and even the conformation of fatty acid synthetase itself has been shown to be light-dependent (Sedgwick, 1973; Delo et al., 1971; E r n s t - F o n b e r g and Bloch, 1971). Anthozoans, including those which lack zooxanthellae, have been shown to contain significant lipid energy reserves, principally in the form of triglycerides and wax esters. Wax esters, in
191
particular, have recently been reported to be major lipid components of a wide variety of marine organisms and to play important roles in the energy budgets of marine food webs (Lee et al., 1970, 1971a, b; Nevenzel, 1970; Benson and Muscatine, 1974; Benson and Lee, 1975). Anthopleura elegantissima contains approximately equal amounts of triglyceride and wax esters (Table 3). This is also the c a s e for several other anemones (Bergmann, 1956; Hoeper and Ackman, 1971), indicating that each are important energy sources in these organisms. Although isolated zooxanthellae also contain about equal amounts of each component, the lower total concentration and the low levels of algal mass in whole anemones makes it reasonable to conclude that the triglyceride and wax ester levels of whole anemones predominantly occur in the animal cells. This has been demonstrated for the hard coral Pocillopora capitata (Patton et al., 1977). The proposal that algae may play a significant role in supplying their hosts with lipid energy is enhanced by the dramatic shift in acetate incorporation from p r e d o m i n a n t l y polar lipid under dark conditions to triglyceride and wax ester during periods of photosynthetic activity (Fig. I). Of particular note is the almost total shift to wax ester synthesis, as opposed to sterol ester synthesis in the light (see "Results"). The greater incorporation of labeled acetate into whole animal triglyceride as opposed to wax ester may be accounted for in two ways. Since little incorporation occurs in zooxanthellae wax esters, only triglycerides may be translocated from algae to animal. If, on the other hand, translocated products are in the form of free fatty acids and/or alcohols, the animal's metabolic pathways favor triglyceride formation. Sargent et al. (1974) and Sargent and M c I n t o s h (1974) have shown that, in copepods, wax ester biosynthesis is determined by the levels of fatty alcohol rather than fatty acid. They also have shown that triglyceride biosynthesis is inhibited by high levels of free fatty alcohol. If the same is true for Anthopleura elegantissima, it would argue for the case that the translocated algal product is predominantly fatty acid. Since increased wax ester synthesis also occurs under light conditions, albeit at a lower level, either lesser amounts of free alcohols are translocated or fatty acids are reduced to fatty alcohols by animal enzyme systems. Acetate incorporation patterns for individual fatty acids suggest that if free fatty acids are translocated from
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a l g a e to a n i m a l , t h e s e a c i d s a r e p r i m a r i l y in the f o r m of s a t u r a t e d or m o n o e n o i c a c i d s , 18 c a r b o n a t o m s or less in length. For isolated zooxanthellae, no appreciable l a b e l w a s f o u n d in f a t t y a c i d s of g r e a t e r t h a n 18 c a r b o n s , or possessing more than three double bonds, e v e n a f t e r 12 h l i g h t i n c u b a t i o n w i t h acetate (Table 4). N o l a b e l e d f a t t y acids higher than octadecenoic (18:1) a c i d w e r e f o u n d in w h o l e a n e m o n e s u n d e r any experimental conditions (Table 5). Short-term s t u d i e s s u c h as t h e s e s h o w l i t t l e s y n t h e s i s a n d t r a n s f e r of a n i m a l e s s e n t i a l f a t t y a c i d s s u c h as l i n o l e i c or l i n o l e n i c f r o m z o o x a n t h e l l a e to a n e m ones. It m a y w e l l be, h o w e v e r , t h a t o v e r longer periods algae serve this function, The interpretation of t h e r e l a t i v e specific activities for i n d i v i d u a l f a t t y a c i d s is d i f f i c u l t , since the values w o u l d d e p e n d on the r a t e s of de novo s y n t h e s i s f r o m a c e t a t e a n d e l o n g a t i o n of pre-existing unlabeled precursors, both of w h i c h a r e u n k n o w n . T h e s e d a t a p r e sent, h o w e v e r , s o m e i n t e r e s t i n g aspects. F o r a l g a e i n c u b a t e d in t h e dark, a t e n f o l d i n c r e a s e in t h e r e l a t i v e s p e c i f ic a c t i v i t y of 16:1 a c i d s o v e r 16:0 a c i d s is n o t e d . A l s o , o v e r 50% of t h e total radioactivity is f o u n d in t h i s acid. T h u s 16:1 f a t t y a c i d s w o u l d b e synthesized de novo r a t h e r t h a n a r i s i n g primarily from desaturation of preexisting unlabeled palmitate. The lack of d e t e c t a b l e a c t i v i t y in 18:0 a c i d s w o u l d f a v o r the a r g u m e n t t h a t 18:1 a c i d s arise from elongation of 16:1 a c i d s . This interpretation is e n h a n c e d b y t h e s h i f t in t h e p e r c e n t a g e of r a d i o a c t i v e incorporation in 16:0, 16:1 a n d 18:1 a c i d s s e e n in l i g h t - i n c u b a t e d a l g a e (Tab l e 4). Here, t h e p e r c e n t i n c o r p o r a t i o n for 16:1 a c i d s is m a r k e d l y r e d u c e d w h i l e t h a t for 18:1 a c i d g r e a t l y i n c r e a s e s . T h e r e l a t i v e s p e c i f i c a c t i v i t y of 18:1 acids also markedly increases over darki n c u b a t e d v a l u e s . T h u s , an a p p a r e n t b l o c k f o r 16:1 c o n v e r s i o n to 18:1 f a t t y a c i d s e x i s t s in t h e a b s e n c e of p h o t o s y n thesis. Another interesting a s p e c t of t h i s s c h e m e w o u l d be t h e p o s i t i o n of t h e d o u b l e b o n d . If t h e p o s i t i o n is t h e u s u a l 18:1, A 9 f o u n d in o l e i c acid, then the hexadecenoic a c i d f o r m e d in t h e a l g a e w o u l d be 16:1, A 7. T h e s e 16:1 A 7 a c i d s h a v e b e e n r e p o r t e d for Dictyostelium discoideum ( D a v i d o f f a n d K o r n , 1 9 6 3 a , b ) , b u t in s m a l l q u a n t i t i e s . More likely would be the formation of vaccenic acid (18:1, A 11) f r o m p a l m i t o l e i c a c i d (16:1,
A 9). W i t h r e s p e c t to w h o l e a n e m o n e s , of n o t e is the a p p a r e n t l a c k of b i o s y n t h e sis of u n s a t u r a t e d fatty acids under
dark conditions (Table 5). T h e i n c r e a s e d l e v e l s of i n c o r p o r a t i o n into monoenoic a c i d s (16:1 a n d 18:1) in t h e l i g h t p o i n t s t o w a r d s an a l g a l c o n t r i b u t i o n to this process, possibly by supplying the 02 a n d N A D P H n e c e s s a r y for the desaturation process. The possibility also exi s t s that, in s o m e way, l i g h t e n h a n c e s the translocation of m o n o e n o i c acids f r o m a l g a l to a n i m a l c e l l s . As noted previously, endozoic algae h a v e b e e n s h o w n to fix a n i m a l - g e n e r a t e d CO 2 a n d to t r a n s l o c a t e organic products, p r i m a r i l y as c a r b o h y d r a t e , to t h e i r a n i m a l h o s t s . It n o w a p p e a r s t h a t z o o x a n t h e l l a e c a n s u p p l y , or e n h a n c e t h e p r o d u c t i o n of a n i m a l l i p i d e n e r g y r e s e r v e s in the f o r m of t r i g l y c e r i d e s a n d w a x esters. Z o o x a n t h e l l a e may thus provide their hosts with a full range of energy stores at low metabolic cost. W h i l e t h e a m o u n t m a y v a r y f r o m s p e c i e s to s p e c i e s , such a relationship w o u l d b e of i m m e n s e survival value and enable such organi s m s to c o l o n i z e or i n h a b i t n u t r i e n t poor environments. Acknowledgements. The authors wish to thank the Department of Chemistry, Georgetown University and the Biochemistry Division, Laboratory of Nuclear Medicine and Radiation Biology, University of California at Los Angeles, for allowing the use of their facilities and equipment for portions of this work.
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Jttne 15, 1979. Communicated by I. Morris,
West Boothbay Harbor